1. Field of the Invention
This invention relates to microfluidic devices having multiple microfluidic channels through which reaction materials flow and, more specifically, to microfluidic devices having features for effecting optical and thermal isolation of microfluidic channels in microfluidic devices.
2. Description of Related Art
The detection of nucleic acids is central to medicine, forensic science, industrial processing, crop and animal breeding, and many other fields. The ability to detect disease conditions (e.g., cancer), infectious organisms (e.g., HIV), genetic lineage, genetic markers, and the like, is ubiquitous technology for disease diagnosis and prognosis, marker assisted selection, correct identification of crime scene features, the ability to propagate industrial organisms and many other techniques. Determination of the integrity of a nucleic acid of interest can be relevant to the pathology of an infection or cancer. One of the most powerful and basic technologies to detect small quantities of nucleic acids is to replicate some or all of a nucleic acid sequence many times, and then analyze the amplification products. Polymerase Chain Reaction (“PCR”) is perhaps the most well-known of a number of different amplification techniques.
PCR is a powerful technique for amplifying short sections of DNA. With PCR, one can quickly produce millions of copies of DNA starting from a single template DNA molecule. PCR includes a three phase temperature cycle of denaturation of DNA into single strands, annealing of primers to the denatured strands, and extension of the primers by a thermostable DNA polymerase enzyme. This cycle is repeated so that there are enough copies to be detected and analyzed. In principle, each cycle of PCR could double the number of copies. In practice, the multiplication achieved after each cycle is always less than 2. Furthermore, as PCR cycling continues, the buildup of amplified DNA products eventually ceases as the concentrations of required reactants diminish. For general details concerning PCR, see Sambrook and Russell, Molecular Cloning—A Laboratory Manual(3rd Ed.), Vols. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (2000); Current Protocols in Molecular Biology, F. M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (supplemented through 2005) and PCR Protocols A Guide to Methods and Applications, M. A. Innis et al., eds., Academic Press Inc. San Diego, Calif. (1990).
Real-time PCR refers to a growing set of techniques in which one measures the buildup of amplified DNA products as the reaction progresses, typically once per PCR cycle. Monitoring the accumulation of products over time allows one to determine the efficiency of the reaction, as well as to estimate the initial concentration of DNA template molecules. For general details concerning real-time PCR, see Real-Time PCR: An Essential Guide, K. Edwards et al., eds., Horizon Bioscience, Norwich, U.K. (2004).
A number of commercial instruments exist that perform real-time PCR. Examples of available instruments include the Applied Biosystems PRISM 7500, the Bio-Rad iCylcer, and the Roche Diagnostics LightCycler 2.0.
More recently, a number of high throughput approaches to performing PCR and other amplification reactions have been developed, for example, involving amplification reactions in microfluidic devices, as well as methods for detecting and analyzing amplified nucleic acids in or on the devices. Thermal cycling of the sample for amplification in microfluidic devices is usually accomplished in one of two methods. In the first method, the sample solution is loaded into the device and the temperature is cycled in time, much like a conventional PCR instrument. In the second method, the sample solution is pumped continuously through spatially varying temperature zones. See, e.g., Lagally et al. (Analytical Chemistry 73:565-570 (2001)), Kopp et al. (Science 280:1046-1048 (1998)), Park et al. (Analytical Chemistry 75:6029-6033 (2003)), Hahn et al. (WO 2005/075683), Enzelberger et al. (U.S. Pat. No. 6,960,437) and Knapp et al. (U.S. Patent Application Publication No. 2005/0042639).
Microfluidic chips are being developed for “lab-on-a-chip” devices to perform in-vitro diagnostic testing. The largest growth area is in molecular biology where DNA amplification is performed in the sealed channels of the chip. Optical detection devices are commonly used to measure the increasing amplicon product over time (Real Time PCR) or to perform a thermal melt to identify the presence of a specific genotype (High Resolution Thermal Melt). Exemplary disclosures related to the imaging of a microfluidic chip to measure the fluorescent product can be found in commonly-owned U.S. application Ser. No. 11/505,358 to Hasson et al. entitled “Real-Time PCR in Micro Channels” (U.S. Pat. Pub. 2008-0003588) and U.S. application Ser. No. 11/606,204 to Hasson et al. entitled “Systems and Methods for Monitoring the Amplification and Dissociation Behavior of DNA Molecules” (U.S. Pat. Pub. 2008-0003594), the respective disclosures of which are hereby incorporated by reference.
A general trend in in-vitro diagnostic microfluidic chips is to make them smaller to conserve sample volumes, material cost, biohazard waste volume, and to reduce thermal mass of the chip for faster PCR cycling. The down side of this size reduction, however, is the increased difficulty in isolating fluidic channels—both thermally and optically—from each other.
Thermal separation of microfluidic channels is a more significant issue if the design has individual heaters for independent thermal cycling in each channel. Independent thermal cycling does not necessarily mean that each channel is driven on a different cycle, which is the extreme of control systems. Even if temperature cycling is performed in unison (i.e., simultaneously in all channels), if there is a separate, dedicated heating element for each channel, there is a potential thermal isolation issue. When one channel is higher or lower than a setpoint temperature, the control system needs to compensate locally. Deviation from setpoint temperature could be caused by varying wattages due to the heating element manufacturing tolerances or by greater heat loss which can occur at the edges of the microfluidic chip, thereby causing lower temperatures in channels adjacent to the edge of the chip. The response to changing inputs to individual heater elements must be isolated in order to create an effective closed loop control. If a significant portion of energy from surrounding heater elements conducts into or out of the area of the heating element for which control input is being adjusted, the response will be hard to predict because of the surrounding influences. The control algorithms may be able to compensate if the surrounding influences were steady state, but in this case they are also being actively controlled. This makes each channel less predictable and may make the system unstable. Improving channel-to-channel isolation by reducing or preventing thermal crosstalk between adjacent channels will improve control of the individual channel temperatures.
Optical isolation of the microfluidic channels is also a problem for some detection systems. As the size of microfluidic chips shrinks, the fluid volume within each channels is reduced, which consequently reduces emission signal levels. Also, as channels through which fluorescent indicators flow are also moved closer together this promotes emission signal crosstalk between adjacent channels. The clear glass or polymer microfluidic chip creates light pipe paths between channels which enables greater crosstalk.
When all channels are illuminated simultaneously, emitted fluorescent light intensity is the critical parameter for detection. Even though the excitation light is basically unidirectional from a single source, the emission light is omni-directional. It is possible, therefore, for one channel to have no emission and light emitted from adjacent channels crosses into the non-emitting one and through particle scattering or refraction at the walls, some light is turned toward the detector. This can elevate signal levels or give false signals.
Also, being omni-directional, most of the emission light does not propagate in the direction of the detector. Much of the light is transmitted through the surface of the micro chip opposite the detector. Accordingly, the intensity of the emission light that actually reaches the detector can be very small, making accurate measurements problematic.
This phenomenon is illustrated in FIGS. 1 and 2. FIG. 1 shows a schematic representation of a partial transverse cross-section of a microfluidic chip 10 having a number of microfluidic channels 16, 18, 20 formed therein. Incident excitation light, represented by arrows 22, is directed through the incident surface 12 at channel 18, light passing through channel 18 toward an opposite surface 14 of the chip 10 is represented by arrows 24, and light reflected or refracted toward adjacent channels 16, 20 is represented by arrows 26, 28, respectively. As shown in FIG. 2, emission light from the channel 18 at which the excitation light 22 is directed is represented by arrows 30. A portion of the emission light 30 is directed at the adjacent channels 16 and 20, as represented by arrows 32 and 34, respectively. The light 26, 32 incident on channel 16 and the light 28, 34 incident on channel 20 may cause emissions from channels 16 and 20, as represented by arrows 36 and 38, respectively. A portion of the emission light 36, 38 from the adjacent channels 16, 20, respectively, will be directed toward the incident surface 12, as represented by arrows 40 and 42, toward the detector (not shown), thereby causing an inaccurate signal strength signal from the channel 18 and/or false signals from the adjacent channels 16, 20.
Also, much of the emission light 30 from the channel 18 will be directed toward surface 14 and will not be detected by the detector that is above surface 12, thus resulting in a relatively weak emission signal from channel 18.
Accordingly, a need exists for a microfluidic chip having means for preventing thermal and optical cross-talk between adjacent microfluidic channels and further for capturing more of the omni-directional emission light from a channel.